Stufies on the Biochemical and Genetic Bases of the Multiplicity of Cytochrome P-450

多种形式的细胞色素P-450存在于肝微粒体中，并且向动物施用各种药物导致诱导细胞色素P-450的特定形式。本项目旨在阐明哺乳动物细胞色素P-450多样性的生化和遗传基础。本项目取得的主要成果可概括如下。首先，从未处理和各种药物处理的兔肝微粒体中纯化了15种形式的细胞色素P-450，并详细检查了它们的性质。第二，从大鼠和兔肝微粒体中分离出16个编码不同形式细胞色素P-450的cDNA克隆，并测定了它们的核苷酸序列，其中包括8个全长克隆。将它们的一级结构与其他实验室测定的一级结构进行比较，得出结论：哺乳动物P-450基因超家族至少由5个家族组成，有些家族还可进一步分为几个亚家族。此外，微观异质性可以检测到的主要苯巴比妥诱导的形式在兔肝脏。第三，克隆了4个大鼠P-450基因、1个兔P-450基因和2个人肾上腺皮质P-450基因，并进行了结构鉴定。在大鼠P-450 c基因的5'上游区域检测到三个调控其表达的元件，其中一个被鉴定为参与药物介导表达的增强子。最后，大鼠P-450 d和兔P-450在酵母细胞中成功表达。发现从酵母细胞中分离的P-450蛋白显示出相对广泛的底物特异性，尽管这些P-450蛋白在一级结构上是同质的。

Multiple forms of cytochrome P-450 occur in liver microsomes and administration of various drugs to animals leads to the induction of a specific form or forms of cytochrome P-450. This project was undertaken to elucidate the biochemical and genetic bases of this multiplicity of mammalian cytochrome P-450. The major results obtained in this project may be summarized as follows. First, 15 forms of cytochrome P-450 were purified from liver microsomes of untreated and variously drug-treated rabbits and their properties were examined in detail. Secondly, 16 cDNA clones including 8 full-length ones coding for different forms of rat and rabbit liver microsomal cytochrome P-450 were isolated and their nucleotide sequences determined. Comparison of their deduced primary structures and those determined in other laboratories leads to the conclusion that the mammalian P-450 gene superfamily consists of at least 5 families and some of the families are further classified into several subfamilies. Furthermore, microheterogeneity can be detected in the major phenobarbital-inducible form in rabbit liver. Thirdly, 4 rat P-450 genes, 1 rabbit P-450 gene and 2 human adrenal cortex P-450 genes were cloned and their structures were determined. In the rat P-450c gene three elements regulating its expression were detected in its 5' upstream region and one of them was identified as an enhancer involved in drug-mediated expression. Finally, Rat P-450d and a rabbit P-450 were successfully expressed in yeast cells. The P-450 proteins thus isolated from yeast cells were found to show relatively broad substrate specificities in spite of the fact that these P-450 proteins are homogeneous with respect to primary structure.